Preyssler-type phosphotungstate is a new family of negative-staining reagents for the TEM observation of viruses

Transmission electron microscopy (TEM) is an essential method in virology because it allows for direct visualization of virus morphology at a nanometer scale. Negative staining to coat virions with heavy metal ions must be performed before TEM observations to achieve sufficient contrast. Herein, we report that potassium salts of Preyssler-type phosphotungstates (K(15-n)[P5W30O110Mn+], M = Na+, Ca2+, Ce3+, Eu3+, Bi3+, or Y3+) are high-performance negative staining reagents. Additionally, we compare the staining abilities of these salts to those of uranyl acetate and Keggin-type phosphotungstate. The potassium salt of Preyssler-type phosphotungstates has the advantage of not requiring prior neutralization because it is a neutral compound. Moreover, the potassium counter-cation can be protonated by a reaction with H+-resin, allowing easy exchange of protons with other cations by acid–base reaction. Therefore, the counter-cations can be changed. Encapsulated cations can also be exchanged, and clear TEM images were obtained using Preyssler-type compounds with different encapsulated cations. Preyssler-type phosphotungstates may be superior negative staining reagents for observing virus. Polyoxotungstates (tungsten-oxide molecules with diverse molecular structures and properties) are thus promising tools to develop negative staining reagents for TEM observations.

www.nature.com/scientificreports/ tetragonal PO 4 unit surrounded by 12 octahedral WO 6 units with T d symmetry (Fig. 2a) 12 . Keggin-type PTA is a highly acidic compound and is mainly used after neutralization with NaOH or KOH [6][7][8][9] . TEM images obtained using this PTA are less clear than those obtained using uranyl acetate. Therefore, other tungsten reagents such as sodium silicotungstate and methylamine tungstate 13 have been considered as alternative negative-staining reagents 7 . These tungsten reagents, along with Keggin-type phosphotungstate, belong to the class of polyoxotungstates, which are anionic tungsten oxide clusters 12 . Polyoxotungstates have diverse molecular structures and physicochemical properties such as stability, solubility, acidity, and crystallinity. Based on this information, we examined a new high-performance negative staining reagent using members of this family of compounds.

Results and discussion
Comparison of negative-staining reagents. Figure 3 shows the TEM images of T4 phages obtained using two common negative staining reagents, uranyl acetate and neutralized Keggin-type PTA, and the potassium salt of Na-encapsulated Preyssler-type phosphotungstate ((K 14 [P 5 W 30 O 110 Na]) as negative staining reagents. Enterobacteria phage T4 (family Myoviridae) was selected as a model virus because its detailed morphology has been established 16 . The T4 phage is constructed from a head with an elongated icosahedron shape, tail part, and base plate. In addition, it has six whisker-like short fibers and long tail fibers (Fig. 3a) 17 . Although the head and tail part were clearly visible using uranyl acetate, the short and long fibers were not observed (Fig. 3b,c), which are similar to reported TEM images [18][19][20][21] . The head, tail part, and long tail fibers were observable using the neutralized Keggin-type PTA; however, the background was not homogeneous (Fig. 3d). In contrast, the background, head, tail part, and long tail fibers were all clearly observed using the potassium salt of Na-encapsulated Preyssler-type phosphotungstate (Fig. 3e,f). It has been reported that the long tail fibers were also observed by using uranyl acetate 17,22 . However, it is worth to note that we could observe clear images without radioactive uranyl acetate.  Fig. S3). From the other 40% of the grid, we obtained images categorized as image B, in which staining reagents coated the T4 phages and parts of the carbon film. In image B, no long tail fibers were observed. However, instances of staining reagents coating T4 phages and clear phage images from only a few percent of the grid were obtained (categorized as image C). In image C, the long tail fibers were clearly observable. The concentration of Preyssler compound in the staining solution was directly proportional to the area where image A was observed and inversely proportional to the areas of images B and C. Moreover, the area ratio of image C reached a maximum when the concentration was 0.3 wt%. However, an area with low contrast was also observed (categorized as image D) that was inversely proportional to the concentration. Furthermore, an AFM image obtained from image area C on the TEM grid ( Fig. 3g and Supplementary  Fig. S5) after TEM observation revealed head and tails. The observed length (230 and 225 nm) of the T4 body (head and tail together) was close to that expected for a T4 phage (Fig. 3a). However, the observed height of the head (47 nm) ( Supplementary Fig. S5) was less than its thickness (78 nm in Fig. 3a), indicating that the head shrank under TEM vacuum conditions.
In the case of the neutralized Keggin-type PTA ( Supplementary Fig. S4), no crystal formation was observed in the high-concentration samples. Although decreasing the concentration improved the images, better images were obtained using Preyssler-type phosphotungstate.

Difference between Preyssler-type and Keggin-type phosphotungstates. Keggin-type and
Preyssler-type phosphotungstates exhibit greatly different stabilities in neutral solution. Supplementary Fig. S6 shows the pH titration curves of Keggin-type PTA and Na-encapsulated Preyssler-type PTA in aqueous solution. For Preyssler-type PTA, the pH rapidly increased when equal moles of NaOH were added to 14 protons, indicating that the Preyssler-type phosphotungstate was stable in aqueous solution with a pH range of 1-12. The reaction in question is as follows: In contrast, the Keggin-type PTA solution remained acidic (pH almost unchanged) after adding equal moles of NaOH to 3 acidic protons. This result indicates that the Keggin-type phosphotungstate molecule ([PW 12 O 40 ] 3− ) was decomposed. Moreover, it has been reported that [PW 12 O 40 ] 3− is stable only under very acidic conditions (pH < 2), and its neutralization produces a complex mixture of phosphotungstate and tungstate species depending on the solution pH 23,24 . In an aqueous solution of pH 7, the main phosphotungstate species detected by phosphorus-31 nuclear magnetic resonance ( 31 P NMR) was mono-defective (lacunary) phosphotungstate ([PW 11 O 39 ] 7− ) ( Supplementary Fig. S2) These species may produce an inhomogeneous background. In contrast, the Preyssler-type phosphotungstate molecule is stable over a wide range (pH 1-12), which might be attributable to the homogeneous background.  Fig. S7). However, long tail fibers were not obtained using tetrabutylammonium ((Bu 4 N) 14 Fig. S7).

Further advantages of Preyssler-type compounds. Preyssler
Furthermore, the encapsulated Na + is exchangeable with other cations that have different charges, such as Ca 2+ , Bi 3+ , Y 3+ , and lanthanoid cations. Such exchange alters the negative charge of the Preyssler molecule without affecting its shape. The change in the negative charge affects the crystallinity of Preyssler molecules and their interaction with the virus surface and carbon film support, changing the performance of the negative staining reagent. Clear TEM images were obtained using these Preyssler-type compounds with different encapsulated cations, such as Ca 2+25 , Y 3+25 , Bi 3+26 , Ce 3+27 , and Eu 3+25 (Supplementary Fig. S8). The Eu 3+ -encapsulated compound (K 12 [P 5 W 30 O 110 Eu(H 2 O)]) was the best negative staining reagent among these compounds (Fig. 4) and clear images were obtained from more than 75 area% of grid ( Supplementary Fig. S8g).
Staining performance with other viruses. The Preyssler-type phosphotungstate was a good negative staining reagent for T4 and other phages examined in the present study. The lambda phage (family Siphoviridae) has an icosahedral head with a diameter of ca. 60 nm, a long flexible tail with a length of ~ 150 nm, a short terminal fiber, and four tail fibers 17,28 , which were all observed (Fig. 5). The T7 phage (family Podoviridae) has an icosahedral head with a diameter of ~ 60 nm, a short tail, and six short fibers 17,29 , which were clearly visible (Fig. 6).

Conclusions
Negative staining has been widely used to observe the morphologies of viruses 6 , other biological particles, lipid vesicles, micelles, liposomes, and polymer particles 30 . Our results indicate that Preyssler-type phosphotungstates are good negative staining reagents for virus observations. Furthermore, tungsten forms a variety of metal oxide clusters known as polyoxotungstate in an aqueous solution, depending on the other elements present and pH 12 .
Polyoxotungstates are promising tools for developing negative staining reagents for TEM observations. Virus observation. For TEM analysis, the phage solution (10 11 PFU mL −1 , 5 μL) was placed in contact with a glow-discharged (JEOL HDT-400, Tokyo, Japan) carbon-coated collodion film on a Cu grid (Nisshin EM, Tokyo, Japan) for 3 min. Excess solution was removed using a filter paper. Subsequently, a drop (5 μL) of staining solution was placed on the grid for 3 min. The staining solution was removed using a filter paper, and the grid was air-dried. TEM (JEOL, JEM-1200EX) with a tungsten filament was employed at 80 kV. The sample grid prepared for the TEM observation was fixed using a carbon tape on a sample holder and observed using SEM (S-4800, Hitachi, Tokyo, Japan) and AFM (SPM-9600, Shimadzu, Kyoto, Japan).

Data availability
All supporting data are found in the supplementary information.